Hepatitis B viral DNA has been found to be a circular DNA which is double stranded over 50 to 85% of its length and 15 to 50% is single stranded in different molecules. The DNA consists of a long DNA strand of constant length (3,200 nucleotides) and a short strand of variable length (1,700 to 2,800 nucleotides). The 5' end of the short strand has been found at a unique site in the molecule and the 3' end varies in location in in different molecules accounting for the single stranded gap of variable length. A DNA polymerase activity in hepatitis B virions use the 3' end of the short strand as primer and the single stranded region of the long strand as template, and the single stranded gaps are closed to make fully double stranded DNA molecules of 3,200 nucleotide pairs (np). The long strand contains a discontinuity or nick approximately 260 np from the 5' end of the short strand. This 260 nucleotide base paired region in circular molecules can be selectively heat denatured to produce linear molecules with single stranded tails on each end. The unique physical features have been localized in a restriction endonuclease cleavage map of the DNA of HBV/adw. Certain restriction sites differ in DNAs of HBV of HBsAg subtypes adw, ayw, and adr. HBV DNA/adw has been cloned in E-coli and DNA of multiple clones examined for base sequence heterogeneity. A tissue culture cell line from a human hepatoma which produces HBsAg has been shown to contain 5 copies per cell of the complete viral DNA sequence integrated at two different sites in cell DNA.